Age production inhibitor

ABSTRACT

Disclosed is an AGE production inhibitor or the like, which effectively inhibits the production of an advanced glycation end product (AGE), while having improved biological safety. Specifically disclosed is an AGE production inhibitor or the like, which contains en extract of cherry tree (preferably blossoms or leaves thereof) and/or a processed product of the extract as an active substance. The AGE production inhibitor or the like contains, as an active substance, at least one compound that is selected from among 1-O-(E)-Caffeoyl-β-D-glucopyranoside, 1-O-(E)-Coumaroyl-β-D-glucopyranoside, 1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol 3-O-β-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol 3-O-(6″-malony)-β-D-glucopyranoside, and Quercetin 3-O-(6″-malony)-β-D-glucopyranoside.

TECHNICAL FIELD

This invention relates to a new AGE production inhibitor. Regarding thisinvention, the AGE production inhibitor has an inhibitory effect onfibroblast apoptosis which is induced by CML-collagen (carboxymethyllysine-collagen), an AGE, and is widely used as a material for foods,medicines, cosmetics or the like.

TECHNICAL BACKGROUND

There may still be much unknown about the production process of AdvancedGlycation End-products (hereinafter referred to as AGE). However, it isthought that the key factor of AGE is a non-enzymatic glycation. Inother words, it is thought that in the initial-stage reaction of AGEproduction, an amino group that exists in protein and an aldehyde groupwhich is a reducing sugar such as glucose or the like, non-enzymaticallyreact to each other (i.e. glycation), which producesAmadori-rearrangement products through Schiff's base. In the late-stagereaction, AGE is produced after a long period of complex cleavages andcondensations.

Diabetes complication includes retinopathy, nephropathia, neuropathy,ischemic cardiac disease, cerebrovascular disease and others. It isknown that one of the factors for the cause of these diseases relates toAGE produced in a hyperglycemic biological body. (For instance, refer tonon-patent reference 1)

Diabetic nephropathy is renal microvascular damage caused by diabetes.Its basic pathological change includes the thickening of the glomerularbasement membrane and the expansion of the mesangial area. Recently anumber of diabetic patients stricken with the end-stage renal disease,caused by diabetic nephropathy increases and so were treated withdialysis. It is thought that AGE which is produced by a continuous stateof hyperglycemia is involved in the development of nephropathy which isdeveloped by 1. enhanced vascular permeability, 2. enhanced depositionof protein and lipoprotein, 3. inactivation of nitric oxide (NO), and 4.accelerated production of extracellular matrix. (See Non-patentReference 2.)

For the sake of the prevention of and for the improvement of diabeticcomplications, various types of AGE production inhibitors have beenresearched and developed.

For example, it is shown that AGE production that induces diabeticcomplications such as nephropathia, retinopathy and others is inhibitedby synthetic drugs (synthetic compounds). (See Patent References 1 and2.) However, such synthetic drugs [that inhibit such] AGE productionremains in the developmental stage, and such synthetic drugs oftenproduce unanticipated side effects.

Contrarily, research is being done of substances or food compositionswhich prevent, control, improve and cure diseases through a dailydietary way of life, instead of using synthetic drugs. Patent References3 to 5, for instance, show compositions of extracts of plants used forfood, which inhibit AGE production.

However, AGE inhibitory activity of such extracts derived from plants isgenerally very low, compared to that of synthetic drugs (syntheticcompounds), and the amount of the extracted active substance isextremely small.

To prevent or cure diabetic complications, continuous and long-termtherapy is required. Thus, it is desired to develop an AGE productioninhibitor that both effectively inhibits AGE production and avoids sideeffects. However, little is known about conventional botanical extractswhich meet public expectation.

In recent years, it has been disclosed that AGE relates closely not onlyto diabetes but also to the aging of the skin. AGE increases with aging,and when Maillard reaction occurs in the collagen part [of the skin]which is the skin protein, the carbonyl group of glucide,non-enzymatically reacts with either the amino group of lysine residueor with the guanidyl group or arginine group in the protein to produceAGE, thus resulting in collagen cross-linking. Once the link structureis formed, the molecule is hardened, and inherent elasticity of the skinis lost. Also, such crosslink substances are treated as foreignsubstances, and the volume of secretion of degrading enzyme(collagenase, elastase) increases, which decreases skin tone andfirmness and weakens the skin, thus resulting in wrinkles, slackness anddullness of the skin.

As such, AGE is now recognized as a trigger of diabetes complications.From the viewpoint of anti-aging care, the production mechanism of AGEis also focused.

PRIOR ARTS Patent References

-   (Patent Reference 1) JP-A-2000-324629-   (Patent Reference 2) JP-A-2005-170935-   (Patent Reference 3) JP-A-2006-28090-   (Patent Reference 4) JP-A-2005-343842-   (Patent Reference 5) JP-A-2005-320262

Non-Patent References

-   (Non-patent Reference 1) Higasi, T. et. al. (Bio Clinica, Vol. 12-2,    pp. 19-21, 1997)-   (Non-patent Reference 2) Baba, T. et. al. (Bio Clinica, Vol. 12-2,    pp. 39-48, 1997)

SUMMARY OF THE INVENTION Problems to be solved by the invention

In such circumstances, the inventors conducted research on thecomponents and amount of the components contained in the extract and ofthe various plant origins. They focused on “cherry tree” which containsplenty of polyphenol. After conducting various experiments, theyrealized that the extract of cherry blossoms and cherry leaves,especially, contain phenylpropanoid glycoside (caffeoyl glucose,coumaroyl glucose, cinnamoyl glucose) and flavonoid glycoside(kaempferol glucoside, quercetin glucoside, kaempferol malonyl glucose,quercetin malonyl glucose), which effectively inhibits AGE productionand promotes the formation of fibroblast-collagen grating.

Also, the inventors learned of a new physiological activity of cherrytree extract, which effectively inhibits fibroblast apoptosis caused byan AGE, CML-collagen (i.e. Carboxymethyl Lysine-collagen), thusproviding this invention.

This invention is to solve the problems, above, and its purpose iseffectively to inhibit AGE production and to provide a biologically safeAGE production inhibitor for the human body.

Also, another purpose of the invention is to provide an apoptosisinhibitor to inhibit the formation of human fibroblast apoptosis causedby AGE, such as CML-collagen (Carboxymethyl lysine-collagen).

Moreover, another purpose of the invention is to provide a fibroblastcollagen grating formation promoter.

Still another purpose of the invention is to provide an AGE productioninhibitor effectively to inhibit AGE production within the fibroblast.

Means to Resolve the Problems

In light of resolving the above problems, the features of this inventionare as follows.

1. The AGE production inhibitor of this invention contains cherryextract and/or its processed substance as an active substance.

2. The AGE production inhibitor of this invention contains cherryblossom extract and/or its processed substance as an active substance.

3. The AGE production inhibitor of this invention contains cherry leafextract and/or its processed substance as an active substance.

4. The AGE production inhibitor of this invention contains at least onecompound as an active substance selected from among1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-glucopyranoside,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol 3-β-D-glucopyranoside,Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside, and (Quercetin3-O-(6″-malony)-β-D-glucopyranoside.

5. The AGE production inhibitor of this invention contains1-O-(E)-Caffeoyl-β-D-glucopyranoside, as an active substance.

6. The AGE production inhibitor of this invention contains Quercetin3-O-β-D-glucopyranoside as an active substance.

The AGE production inhibitor of this invention, according to the abovefeatures 1 to 6, allows the edible and safe cherry-derived extract toinhibit effectively AGE production and to avoid side effects, thusproviding an ingredient for foods, medicines, cosmetics and otherproducts which are helpful in effectively preventing or treatingdiabetic complications.

7. The apoptosis inhibitor of this invention contains cherry extractand/or its processed substance as an active substance, which inhibitsfibroblast apoptosis caused by CML-collagen (i.e. Carboxymethyllysine-collagen) which is an AGE.

8. The apoptosis inhibitor of this invention contains cherry blossomextract and/or its processed substance as an active substance, whichinhibits fibroblast apoptosis caused by CML-collagen (i.e. Carboxymethyllysine-collagen) which is an AGE.

9. The apoptosis inhibitor of this invention contains cherry leafextract and/or its processed substance as an active substance, whichinhibits fibroblast apoptosis caused by CML-collagen (i.e. Carboxymethyllysine-collagen) which is an AGE.

10. The apoptosis inhibitor of this invention contains at least oneactive substance selected from among1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-glucopyranoside,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol3-O-β-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside and Quercetin3-O-(6″-malony)-β-D-glucopyranoside, which inhibits the fibroblastapoptosis caused by the CML-collagen (i.e. Carboxymethyllysine-collagen) which is an AGE.

11. The apoptosis inhibitor of this invention contains1-O-(E)-Caffeoyl-β-D-glucopyranoside as an active substance, whichinhibits fibroblast apoptosis caused by CML-collagen (i.e. Carboxymethyllysine-collagen) which is an AGE.

12. The apoptosis inhibitor of this invention contains Quercetin3-O-β-D-glucopyranoside and/or Quercetin3-O-(6″-malony)-β-D-glucopyranoside as an active substance, whichinhibits fibroblast apoptosis caused by CML-collagen (i.e. Carboxymethyllysine-collagen) which is an AGE.

The AGE production inhibitor of this invention, according to the abovefeatures 7 to 12, allows cherry-derived extract, which is safe for food,to inhibit effectively the fibroblast apoptosis caused by CML-collagen(Carboxymethyl lysine-collagen), thus providing a material for foods,medicines, cosmetics and other products, which is helpful in effectivelypreventing or inhibiting the skin from aging.

13. The human fibroblast-collagen grating-formation promoter of thisinvention contains cherry-tree extract and/or its processed substance asan active substance.

14. The human fibroblast-collagen grating-formation promoter of thisinvention contains cherry-blossom extract and/or its processed substanceas an active substance.

15. The human fibroblast-collagen grating-formation promoter of thisinvention contains cherry-leaf extract and/or its processed substance asan active substance.

16. The human fibroblast-collagen grating-formation promoter of thisinvention contains at least one active substance selected from amongthis group of compounds: 1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-glucopyranoside,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol3-O-β-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside and Quercetin3-O-(6″-malony)-β-D-glucopyranoside.

17. The human fibroblast-collagen grating-formation promoter of thisinvention contains 1-O-(E)-Caffeoyl-β-D-glucopyranoside as an activesubstance.

18. The human fibroblast-collagen grating-formulation promoter of thisinvention contains Quercetin 3-O-β-D-glucopyranoside as an activesubstance.

The human fibroblast-collagen grating-formulation promoter of thisinvention, according to the above features 13 to 18, allowscherry-derived extract, which is safe for food, to promote effectivelyhuman fibroblast collagen grating formulation, thus providing a materialfor foods, medicines, cosmetics and other products which are helpful ineffectively preventing or inhibiting the skin from aging.

19. The AGE production inhibitor within the fibroblast of this inventioncontains cherry-tree extract and/or its processed substances as activesubstances.

20. The AGE production inhibitor within the fibroblast of this inventioncontains cherry-blossom extract and/or its processed substance as anactive substance.

21. The AGE production inhibitor within the fibroblast of this inventioncontains cherry-leaf extract and/or its processed substance as an activesubstance.

22. The AGE production inhibitor within the fibroblast of this inventioncontains at least one active substance selected from among this group ofcompounds, i.e. 1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-glucopyranoside,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol3-O-β-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside and Quercetin3-O-(6″-malony)-β-D-glucopyranoside.

23. The AGE production inhibitor within the fibroblast of this inventioncontains 1-O-(E)-Caffeoyl-β-D-glucopyranoside as an active substance.

24. The AGE production inhibitor within the fibroblast of this inventioncontains Quercetin 3-O-β-D-glucopyranoside as an active substance.

The AGE production inhibitor within the fibroblast of this invention,according to the above features 19 to 24, allows cherry-derived extract,which is safe for food, to effectively inhibit AGE production within thefibroblast, thus providing a material for foods, medicines, cosmeticsand other products which are helpful in effectively preventing orinhibiting the skin from aging.

25. The cherry-tree extract of this invention holds at least one activesubstance selected from among this group of compounds, i.e.1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-glucopyranoside,1-O-(E)-Cinnamoyl-6-D-glucopyranoside, Kaempferol3-O-β-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside, and Quercetin3-O-(6″-malony)-β-D-glucopyranoside.

26. The cherry-tree extract of this invention holds all of thesubstances of this group of compounds, i.e.1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-glucopyranoside,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol3-O-β-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside, and Quercetin3-O-(6″-malony)-β-D-glucopyranoside.

27. The cherry-tree extract of this invention holds all compounds,1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-glucopyranoside,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol3-O-β-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside, Quercetin3-O-(6″-malony)-β-D-glucopyranoside and said cherry-tree extract isobtained by a manufacturing method having the following three steps:

(a) extracting one or both cherry blossom extract and the cherry leafextract with 20 to 50 wt % hydrous ethanol,(b) absorbing the extract obtained in step (a) by an absorbentcomprising porous synthetic resin, elute the extract in water and removethe eluted extract, and(c) after step (b) further eluting the eluted extract with a loweralcohol content (carbon numbers 1 to 5), and then concentrate thefurther eluted extract.

28. The AGE production inhibitor of this invention holds cherry-treeextract and/or its processed active substance according to the abovefeature 27.

29. The apoptosis inhibitor of this invention holds cherry-tree extractand its processed substance as an active substance, according to theabove feature 27, and inhibits fibroblast apoptosis caused by theCML-collagen (i.e. Carboxymethyl lysine-collagen) which is an AGE.

30. The human fibroblast-collagen grating-formulation promoter of thisinvention holds cherry-tree extract and/or its processed substance as anactive substance according to the above feature 27.

31. The AGE production inhibitor within the fibroblast of this inventionholds cherry-tree extract and/or its processed substance as an activesubstance according to the above feature 27.

32. The agent, to prevent and cure the diabetic complications, of thisinvention holds cherry-tree extract and/or its processed substance as anactive substance according to the above feature 27.

33. The agent, to prevent the skin from aging and to improve it, of thisinvention holds cherry-tree extract and/or its processed substance as anactive substance according to the above feature 27.

The invention, according to the above features 25 to 33, allows thecherry-derived extract, which is safe for food, to inhibit AGEproduction effectively without side effects, thus providing a materialfor foods, medicines, cosmetics and other products which are helpful ineffectively preventing or treating diabetic complications.

The invention, according to the above features 25 to 33, alsoeffectively inhibits fibroblast apoptosis caused by CML-collagen(Carboxymethyl lysine-collagen), thus effectively promoting the humanfibroblast-collagen grating-formulation, thus effectively inhibiting AGEproduction within the fibroblast, thus providing a material for foods,medicines, cosmetics and other products which are helpful in effectivelypreventing the aging of the skin and for improving it.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart explaining the search for the substances contained inthe cherry-blossom extract.

FIG. 2 is a microscopic image of fibroblast caused by CML-collagen(Carboxymethyl lysine-collagen) regarding the cherry blossom extract ofthe example of this invention.

FIG. 3 is a megascopic image of 24 and 48 hours of a culture ofcollagen-grating formulations of fibroblasts glycosylated by glyoxal.

FIG. 4 shows microscopic images (×100) in 24 hours of culture accordingto FIG. 3.

FIG. 5 shows microscopic images (×200) in 48 hours of culture accordingto FIG. 3.

FIG. 6 shows the effect on AGE production (carboxy methyllsin protein),using Western blotting, within the fibroblast (human diploid fibroblast)caused by glyoxal.

FIG. 7 shows the effect on AGE production within the glyoxal-inducedskin, using Western blotting, regarding the cherry blossom extract as anexample of this invention.

FIG. 8 shows microscopic images (×400) of TUNEL positive cells whichidentify the effect on dermal cell apoptosis induced by CML-collagen(Carboxymethyl lysine-collagen), regarding the cherry blossom extract asan example of this invention.

MODELS FOR CARRYING OUT THE INVENTION

Hereinafter, examples of this invention are described. Of thisinvention, the AGE production inhibitor and fibroblast apoptosisinhibitor and human fibroblast-collagen grating-formulation promoter andAGE production inhibitor within the fibroblast and cherry extract(hereinafter collectively referred to as the AGE production inhibitor)contain, as a bioactive substance, at least one substance selected fromamong 1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-glucopyranoside,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol3-O-β-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside and Quercetin3-O-(6″-malony)-β-D-glucopyranoside.

1-O-(E)-Caffeoyl-β-D-glucopyranoside is a compound as described inFormula 1, below.

1-O-(E)-Coumaroyl-3-D-glucopyranoside is a compound as described inFormula 2, below.

1-O-(E)-Cinnamoyl-β-D-glucopyranoside is a compound as described inFormula 3, below.

Kaempferol 3-O-β-D-glucopyranoside is a compound as described in Formula4, below.

Quercetin 3-O-β-D-glucopyranoside is a compound as described in Formula5, below.

Kaempferol 3-O-(6″-malony)-β-D-glucopyranoside is a compound asdescribed in Formula 6, below.

Quercetin 3-O-(6″-malony)-β-D-glucopyranoside is a compound as describedin Formula 7, below.

It is preferable that AGE production inhibitor of this invention containat least the compound 1-O-(E)-Caffeoyl-β-D-glucopyranoside (Formula 1)or Quercetin 3-O-β-D-glucopyranoside (Formula 5), which are selectedfrom among the compounds, as described in Formulae 1 to 7, or especiallythe compound, 1-O-(E)-Caffeoyl-β-D-glucopyranoside (Formula 1).

Any method used in obtaining the compound as described in Formulae 1 to7, above, is not specifically limited. Such a compound is, however,preferably to be extracted from plants, which makes it simple to obtain.Also, when extracting the compound from plants, it is preferable toextract it from the cherry tree.

Cherry blossom (referred to as Sakura in Japanese) is the generic nameof the Genus Cerasus of Rosaceae, excluding apricots and peaches, andrefers to plants belonging to Subgenus Cerasus.

The types of cherry blossoms (Sakura) used of this invention are notspecifically limited. Cherry blossoms of this invention include, forexample, prunus yamazakukra (Cerasus jamasakura), edohigan (Cerasusspachiana Lavalee ex H. Otto var. spachiana forma ascendens (Makino) H.Ohba), mamezakura (Prunus incisa Thunb. ex Murray) choujizakura (Prunusapetara Fr. et Sav.), miyamazakura (Prunus maximowiczii Rupr.),shinamizakura (Prunus pseudo-cerasus Lindl.) and others, but not limitedto these.

The part of the cherry, which is used for the material of thisinvention, is not specifically limited but includes its leaf, stem,trunk, blossom, fruit and other parts. However, it is preferable to usethe cherry leaf or blossom.

If extraction is done by using a polar solvent, the type of such a polarsolvent is not limited but includes water, methanol, ethanol,isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propyleneglycol, glycerin, acetic acid, ethyl acetate, ether, hexane and others.Of these, water, methanol, and ethanol are especially preferable, whichefficiently extracts the active substance. Also, only one or moresolvents from among the polar solvents, above, can be used.

When using water as an extracting solvent, the extraction temperature isto be set between 20 and 100 degrees centigrade, preferably between 40and 70 degrees centigrade. If the temperature is too low, the activesubstance will not efficiently be extracted. On the other hand, if thetemperature is too high, cyanogen compounds contained in the cherry-tree(sakura) will be eluted and remain in the water, and the activesubstance will be degraded in such a high temperature, which is notpreferable, either. The types of water used for the extraction are notspecifically limited but include tap water, distilled water, mineralwater, ionized alkaline water and others.

When using aqueous alcohol as an extracting solvent, the concentrationof alcohol is preferably to be set at 25 to 50 wt % (percent by weight),preferably at 25 to 30 wt %. If the level of alcohol concentration isbelow 20 wt %, it will be hard to get a high level of active substance.Contrarily, if the concentration of alcohol is over 50 wt %, the yieldwill be low, due to impurities or the like.

Also, in case where the concentration of alcohol is 30 wt % or more, theextraction temperature is preferably to be set at 0 to 95 degreescentigrade, more preferably at 0 to 60 degrees centigrade. As for theextraction of aqueous ethanol, it is preferable to repeat theextractions in various concentrations so as to improve the content rateof the active substance.

Also, when conducting an extraction with a polar solvent, the method ofextraction is not specifically limited. The method of the extraction,for instance, includes a continuous extraction, a soaking extraction, acountercurrent extraction or any other extraction which can be used withany optional equipment at room temperature or by heating under reflux.Also, any one, or any combination of the above methods can be used forthe extraction. Furthermore, the extraction can be conducted once ormore than once.

As for the specific extraction method, put the extraction ingredientinto the processing vessel filled with extracting solvent, and stir itso that the active substance seeps into the solvent.

For instance, when using hydrous ethanol as the extracting solvent, theextraction is conducted with the extracting solvent approximately 3 to100 times as much weight as the extraction ingredient, from one to 150minutes. After the active substance seeps into the solvent, filter itand remove the residue to obtain the extracted liquid. After that,according to the ordinary method, apply the dilution, concentration,drying, refining method or the like to the extracted liquid, so as toobtain the extract containing polyphenol, as described in the aboveFormulae 1 to 7 or the like.

Also, the refining method includes, for example, an activated carbontreatment, a resin absorption treatment, a silica gel treatment as wellas another method using ion-exchange resin, and liquid-liquidcountercurrent distribution, and membrane separation, or the like.

In addition, when the extraction is done by supercritical extraction,the supercritical fluid is not specifically limited but includes, forinstance, carbon dioxide, nitrogen or the like, of which one or morefluids can be used at the same time. However, carbon dioxide ispreferable, since it can easily elute the active substance. Also, theknown conventional method of extraction can also be used. After theextraction, according to the ordinary method, apply the dilution,concentration, drying, refining method or the like to the extractedliquid, so as to obtain the extract containing polyphenol, as describedin the above Formulae 1 to 7, or the like.

To obtain the polyphenol, as described in the above Formulae 1 to 7, itis preferable to use the following processes:

(a) extract the cherry blossom extract and/or the cherry leaf extractwith 20 to 50 wt % hydrous ethanol,(b) absorb the extract obtained in Process (a) by absorbent, elute theextract in water and remove the eluted extract, and(c) elute the extract with an aqueous solvent, and then concentrate theeluted extract.

Using the above processes efficiently obtains the extract containing ahigh level of polyphenol, as described in the above Formulae 1 to 7.

As for the absorbent used in the above referenced process (b), one ormore absorbents can be selected and used from among the knownconventional absorbent such as an ion-exchange resin, a syntheticabsorbent resin, an activated carbon, a chelating resin, silica gel, analumina gel series absorbent, a porous glass or the like. It ispreferable to use a column chromatography with porous syntheticabsorbent resin such as DIAION®HP-20 (Mitsubishi Chemical Corporation),SEPABEADS®SP-207 (Mitsubishi Chemical Corporation) or the like.

As for the aqueous solvent used in the above referenced process (c), alower alcohol content (carbon numbers 1 to 5) can be used. Methanoland/or ethanol is especially preferable.

The AGE production inhibitor or the like of this invention effectivelyinhibits AGE production and prevents diabetic complications such asnephropathy, retinopathy or the like. Also, it especially inhibits AGEproduction within the fibroblast to improve the crosslink of thecollagen part of the skin, thus preventing the skin from hardening.Moreover, it inhibits fibroblast apoptosis occurring after theproduction of AGE, thus retaining the elasticity of the skin, thuspreventing flecks and dullness of the skin. Furthermore, it improvescollagen cross-linking after AGE production, thus preventing the skinfrom hardening.

The AGE production inhibitor or the like of this invention can be usedas an ingredient for any food and drink such as, edible oils (saladoils), confectionary (chewing gums, candies, caramels, chocolates,cookies, jellies, gummies, tablet shaped sweets or the like), noodles(Japanese buckwheat noodles called Soba, Japanese wheat noodles calledUdon, Chinese noodles called Ramen or the like), dairy food (milk, icecream, yogurt, or the like), seasoning (fermented bean paste calledMiso, Soy sauce called Shoyu, or the like), soups, drinks (juice,coffee, black tea, green tea, carbonated drink, sports supplement drinksor the like) including general foods and healthy food (tablet type,capsule type or the like), nutritional supplements (nutritioussupplement drink or the like). The AGE production inhibitor or the likeof this invention can be used for the above foods and drinks.

According to the type of the above foods and drinks, the followingingredients can be added: Glucose, fructose, sucrose, maltose, sorbitol,stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid,saccinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodiumerythorbate, glycerin, propylene glycol, glycerin fatty acid ester,polyglycerol fatty acid ester, sucrose fatty acid ester, sorbitan fattyacid ester, propylene glycol fatty acid ester, Arabian gum, carrageenan,casein, gelatin, pectine, agar-agar (gelatin made from seaweed), vitaminB family, nicotinic-acid amide, pantothenate acid calcium, amino acids,calcium salts, pigment, aroma chemicals, preservatives, or the like.

Also, other antioxidants or compounding ingredients of healthy foodinclude the antioxidant “reduced ascorbic acid” or vitamin C and alsothe antioxidants, vitamin E, reduced glutacin, tocotrienol, vitamin Aderivative, lycopene, rutin, β-cryptoxanthin, astaxanthin, zeaxanthin,fucoxanthin, uric acid, ubiquinone, coenzyme Q-10, folic acid, garlicextract, allicin, sesamin, lignans, catechin, isoflavone, chalcone,tannins, flavonoids, coumarin, isocoumarines, blueberry extract,ingredients for healthy food (V. (vitamin) A, V.B1, V.B2, V.B6, V.B12,V.C, V.D, V.E, V.P, choline, niacin, pantothenic acid, calcium folicacid, EPA, oligosaccharide, dietary fiber, squalene, soybean lecithin,taurine, dunalliela, protein, octacosanol, DHA, egg-yolk lecithin,linoleic acid, lactoferrin, magnesium, zinc, chrome, selenium, kalium,hem iron, oyster extract, chitosan, chitin oligosaccharides, collagen,chondroitin, elastin, turmeric, sweetroot, extract of Chinese wolfberryfruit called kukoshi, cinnamon, may, ginger, bracket fungus, shijimiclam (corbicula japonica) extract, snapping turtle, sweetroot, lyciifructus, cinnamomi cortex, hawthorn, plantain, chamomilla, chamomile,dandelion, hibiscus, honey, pollen, royal jelly, lime, lavender, rosehip, rosemary, sage, bifidobacteria, streptococcus faecalis,lactobacillus, wheat germ oil, sesame oil, perilla oil, soybean oil,medium chain fatty acid, agaricus, ginko biloba extract, chondroitin,brown rice germ oil, leechee, onion, DHA, EPA, DPA, rubus suavissimuss.lee, plant worm (cordyceps sineusis saccardo), garlic, larvae of abee, papaya, pu-erh-tea, propolis, Acer nikoense, hericium erinaceum,royal jelly, saw palmetto, hyaluronic acid, gaba, harp seal oil, sharkcartilage, lecithin, phosphatydyl serine, panax notoginseng, mulberryleaf, soybean extract, echinacea purpurea, acanthopanax senticosus,barley extract, olive leaf, olive, gymnema, banaba, salacia reticulata,garcinia, chitosan, saint john's wort, jujube, carrot, passion flower,broccoli, placenta, coix lacryma-bobi, grape seed, peanut skin,bilberry, black cohosh, milk thistle, laurel, sage, rosemary, apocynumvenetum, black vinegar, bitter gourd, maca, carthamus tinctorius,linseed, oolong tea, flower aculeus, caffeine, capsaicin,xylo-oligosaccharide, glucosamine, buckwheat, citrus, dietary fiber,protein, prune, spirulina, young green barley leaf, nucleic acid,natural yeast, shiitake mushroom (lentinus edodes), Japanese plum, aminoacid, extract of deep sea shark, morinda citrifolia, oyster, snappingturtle, champinion, common plantain, acerola, pineapple, banana, peach,apricot, melon, strawberry, raspberry, orange, fucoidan, acer nikoense,cranberry, zinc, iron, ceramide, silk peptide, glycine, niacin, chastetree, ceramide, L-cysteine, red grape juice, millet, horsetail, bition,centrlla asiatica, lonicera caerulea, pycnogenol, petasites japonicus,rhubarb, clove, pu-erh, citric acid, beer yeast, mellilot, black zinger,ginger, curcuma zedoaria, nattokinase, ang-khak (Chinese red rice),tocotrienol, lactoferrin, tartary buckwheat, cocoa, houttuynia cordata,kiwi fruit, piper longum, lotus leaf, pfaffia and star fruit.

A more specific use of the extracting method is herein described.Firstly, spray-dry or freeze-dry the cherry-tree extract with powderedcellulose, then make it a powder, a granule, a tablet, or liquid toeasily use with different kinds of food and drinks (ready-to eat mealsor the like). Also, it is possible to dissolve the cherry-tree extract,for instance, oil and fat, ethanol, glycerin, or a mixture of thesesubstances, and to use such a liquid for dry food or drinks. Also it ispossible to make it into a powder or granule by mixing it with a bindersuch as Arabian gum, dextrin, or the like to add to dry food or drinks.

The total amount of the active substance of the AGE production inhibitoror the like of this invention, which is added to the food and drinks, ispreferably 1 to 20 wt % or less, since the major objective of thisinvention is health maintenance.

The AGE production inhibitor or the like of this invention can be usedas the raw material of medicines (including drugs and quasi-drugs). TheAGE production inhibitor or the like of this invention can beappropriately mixed with raw materials for drug formulations, forinstance, vehicles (glucose, sucrose, white soft sugar, sodium chloride,starch, calcium carbonate, kaolin, crystalline cellulose, cacao oil,hydrogenated vegetable oil, talc, or the like), binders (distilledwater, normal saline solution, ethanol in water, ethanolic solution,simple syrup, dextrose in water, starch solution, gelatin solution,carboxymethyl cellulose, potassium phosphate, polyvinyl pyrrolidone, orthe like), disintegrating agents (alginate sodium, agar-agar, sodiumhydrogen carbonate, sodium lauryl sulphate, stearic acid monoglyceride,starch, lactose, powdered aracia, gelatin, ethanol, or the like),suppressive agents for disintegration (white soft sugar, stearin, cacaooil, hydrogenated oil, or the like), absorption promoters (quaternaryammonium base, sodium lauryl sulphate, or the like), adsorbents(glycerin, starch, lactose, kaolin, bentonite, silic acid, or the like),lubricant agents (purified talc, stearate, polyethyleneglycol, or thelike)

The AGE production inhibitor or the like of this invention can be orallyadministered in the form of tablets, pills, soft or hard capsules,subtle granules, powders, granules, liquids, or the like. However, itcan also be parenterally administered in the different forms of solutionor together with a dispersant, a suspending agent, a stabilizer, or thelike such as a medical skin patch, a lotion, an ointment, a cream or thelike.

The applied dose can be adjusted according to the method ofadministration, the condition of the disease, the age of the patient, orthe like. However, adults can normally take approx. 1 to 1,000 mg of anactive substance per day, while children can take 0.5 to 500 mg per day.The compounding ratio of the AGE production inhibitor or the like ofthis invention can be adjusted according to the mode of administration.When the dietetic composition is orally administered or mucosallyadministered, the applied dose is preferably 0.3 to 15.0 wt %. When thedietetic composition is parenterally administered, the dose ispreferably 0.01 to 10 wt %. The dose varies depending on the conditions.Therefore, a dose which is less than the above-stated amount may besufficient, or a greater amount may sometimes be needed.

The AGE production inhibitor or the like of this invention can be mixedwith cosmetics such as emulsions, soaps, facial cleansers, bath agents,creams, skin lotions, colognes, shaving creams, shaving lotion, beautyoils, tanning lotions, sunscreen lotions, face powders, foundations,perfumes, facial masks, nail creams, nail enamels, nail-polish removers,eyebrow pencils, blushers, eye creams, eye shadows, mascaras, eyeliners, sticks, lip creams, shampoos, hair conditioners, hairdyes,dispersion liquids, cleansing preparations, or the like. Also, the AGEproduction inhibitor or the like of this invention can be mixed withdrugs and quasi-drugs such as ointments, cream pharmaceuticals, liquidsfor external use or the like.

Within the functional range of the AGE production inhibitor or the likeof this invention, the above items for external skin use can be alsomixed with the ingredients of cosmetics, quasi-drugs, or the like. Thoseingredients include, for example, oil, higher alcohol, fatty acids,ultraviolet absorbers, powders, pigments, surface active agents,polyhydric alcohol and sugar, polymers, biologically active ingredients,solvents, antioxidants, aroma chemicals (perfume material), antiseptics.However, those ingredients usable in the present invention are notlimited to these examples.

(1) Specific Examples of Oil Ester-Type Oil Phase Ingredient:

Triglyceryl 2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropylmyristate, butyl myristate, isopropyl palmitate, ethyl stearate, octylpalmitate, isocetyl isostearate, butyl stearate, butyl myristate, ethyllinoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate,isostearyl myristate, isostearyl palmitate, octyldodecyl myristate,isocetyl isostearate, diethyl sebacate, diisopropyl adipate, isoarachylneopentanoate, caprylic-capric acid triglyceride, trimethylolpropanetri-2-ethylhexanoate, trimethylolpropane triisostearate, pentaerythritoltetra-2-ethylhexanoate, cetyl caprylate, decyl laurate, hexyl laurate,decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate,decyl oleate, cetyl ricinoleate, isostearyl laurate, isotridecylmyristate, isocetyl myristate, isostearyl myristate, isocetyl palmitate,isostearyl palmitate, octyl stearate, isocetyl stearate, isodecyloleate, octyldodecyl oleate, octyldodecyl linoleate, isopropylisostearate, cetostearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate,hexyl isostearate, ethylene glycol dioctanoate, ethylene glycoldioleate, propylene glycol dicaprate, propylene glycol di(caprylate/caprate), propylene glycol dicaprylate, neopentyl glycoldicaprate, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryltriundecylate, glyceryl triisopalmitate, glyceryl triisostearate,octyldodecyl neopentanoate, isostearyl octanoate, octyl isononanoate,hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetylisostearate, isostearyl isostearate, octyldecyl isostearate,polyglycerin oleate, polyglycerin isostearate, dipropyl carbonate,dialkyl carbonate (C12-18), triisocetyl citrate, triisoarachyl citrate,triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate,octyldecyl lactate, triethyl citrate, acetyltriethyl citrate,acet-yltributyl citrate, trioctyl citrate, diisostearyl malate,2-ethylhexyl hydroxystearate, 2-ethylhexyl succinate, diisobutyladipate, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate,cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryloleate, dihydrocholesteryl oleate, phytosteryl isostearate, phytosteryloleate, isocetyl 12-stearoylhydroxystearate, stearyl12-stearoylhydroxystearate and isostearyl 12-stearoylhydroxystearate.From among the above ingredients, one only can be used, or two or morecan be used together.

Hydrocarbon-Type Oil Phase Ingredient:

Squalane, liquid paraffin, α-olefin oligomer, isoparaffin, ceresin,paraffin, liquid isoparaffin, polybutene, microcrystalline wax, Vaselineor the like.

Animal and Plant Oil, Hardened Oil Thereof, and Wax of Natural Origin:

Animal oils and hardened oils thereof, such as beef tallow, hardenedbeef tallow, lard, hardened lard, horse oil, hardened horse oil, minkoil, orange roughy oil, fish oil, hardened fish oil and egg yolk oil;plant oils and hardened oils thereof such as avocado oil, almond oil,olive oil, cacao oil, apricot kernel oil, kukui nut oil, sesame oil,wheat germ oil, rice germ oil, rice bran oil, safflower oil, sheabutter, soybean oil, evening primrose oil, perilla oil, tea seed oil,tsubaki oil (camellia japonica oil), corn oil, rapeseed oil, hardenedrapeseed oil, palm kernel oil, hardened palm kernel oil, palm oil,hardened palm oil, peanut oil, hardened peanut oil, castor oil,hydrogenated castor oil, sunflower oil, grape seed oil, jojoba oil,hardened jojoba oil, macadamia nut oil, meadowfoam seed oil, cottonseedoil, hardened cottonseed oil, conoanut oil, hardened cocoanut oil; andwaxes such as beeswax, high acid number beeswax, lanolin, reducedlanolin, hardened lanolin, liquid lanolin, carnauba wax and montan wax.From among the above ingredients, one only can be used, or two or morecan be used together.

Silicone-Type Oil Phase Ingredient:

Dimethylpolysiloxane, methylphenylpolysiloxane, methylcyclopolysiloxane,octamethylpolysiloxane, decamethylpolysiloxane,dodecamethylcyclosiloxane, methylhydrogenpolysiloxane,polyether-modified organopolysiloxane,dimethylsiloxanemethylcetyloxysiloxane copolymer,dimethylsiloxane-methylstearoxysiloxane copolymer, alkyl-modifiedorganopolysiloxane, terminal-modified organopolysiloxane, amino-modifiedsilicone oil, amino-modified organopolysiloxane, dimethiconol, siliconegel, acryl silicone, trimethylsiloxysilicic acid and silicone RTVrubber. From among the above ingredients, one only can be used, or twoor more can be used together.

Fluorine-Type Oil Phase Ingredient:

Perfluoropolyether, fluorine-modified organopolysiloxane, fluorinatedpitch, fluorocarbon, fluoroalcohol andfluoroalkyl-polyoxyalkylene-comodified organopolysiloxane. From amongthe above ingredients, one only can be used, or two or more can be usedtogether.

(2) Specific Examples of Higher Alcohol

Lauryl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol,isostearyl alcohol, oleyl alcohol, behenyl alcohol, 2-ethylhexanol,hexadecyl alcohol and octyl dodecanol. From among the above ingredients,one only can be used, or two or more can be used together.

(3) Specific Examples of Fatty Acid

Caprylic acid, capric acid, undecylenic acid, lauric acid, myristicacid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid,oleic acid, linoleic acid, linolenic acid, arachic acid, arachidonicacid, behenic acid, erucic acid and 2-ethylhexanoic acid. From among theabove ingredients, one only can be used, or two or more can be usedtogether.

(4) Specific Examples of Ultraviolet Absorber

Para-aminobenzoic acid, amyl para-aminobenzoate, ethyldihydroxypropylpara-aminobenzoate, glyceryl para-aminobenzoate, ethylpara-aminobenzoate, octyl para-aminobenzoate, octyldimethylpara-aminobenzoate, ethylene glycol salicylate, octyl salicylate,triethanolamine salicylate, phenyl salicylate, butylphenyl salicylate,benzyl salicylate, homomethyl salicylate, benzyl cinnamate, octylpara-methoxycinnamate, 2-ethylhexyl para-methoxycinnamate, glycerylmono-2-ethyl hexanoate di-para-methoxycinnamate, isopropylpara-methoxycinnamate, diethanolamine para-methoxyhydrocinnamate,diisopropyl diisopropylcinnamic acid ester mixture, urocanic acid, ethylurocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and a salt thereof, dihydroxymethoxybenzophenone, sodiumdihydroxymethoxybenzophenonedisulfonate, dihydroxybenzophenone,dihydroxydimethoxybenzophenone, hydroxyoctoxybenzophenone,tetrahydroxybenzophenone, butylmethoxydibenzoylmethane,2,4,6-trianilino-p-(carbo-2-ethylhexyl-1-oxy)-1,3,5-triazine,2-(2-hydroxy-5-methylphenyl)benzotriazole, methyl-O-aminobenzoate,2-ethylhexyl-2-cyano-3,3-diphenylacrylate, phenylbenzimidazole sulfuricacid, 3-(4-methylbenzylidene)camphor, isopropyldibenzoylmethane,4-(3,4-dimethoxyphenylmethylene)-2,5-doxy-1-imidazolidinepropionate, andpolymer derivatives and silane derivatives thereof. From among the aboveingredients, one only can be used, or two or more can be used together.

(5) Specific Examples of Powder and Pigment

Pigments such as Food Red 104, Food Red 201, Food Yellow 4, Food Blue 1and Food Black 401; lake pigments such as Food Yellow 4 AL lake and FoodYellow 203 BA lake; polymers such as nylon powder, silk powder, urethanepowder, Teflon® powder, silicone powder, polymethyl methacrylate powder,cellulose powder, starch, silicone elastomer spherical powder andpolyethylene powder; color pigments such as yellow iron oxide, red ironoxide, black iron oxide, chromium oxide, carbon black, ultramarine andiron blue; white pigments such as zinc oxide, titanium oxide and ceriumoxide; extender pigments such as talc, mica, sericite, kaolin and platebarium sulfate; pearl pigments such as mica titanium; metal salts suchas barium sulfate, calcium carbonate, magnesium carbonate, aluminumsilicate and magnesium silicate; inorganic powders such as silica andalumina; metal soaps such as aluminum stearate, magnesium stearate, zincpalmitate, zinc myristate, magnesium myristate, zinc laurate and zincundecylenate; bentonite; smectite; and boron nitride. From among theabove ingredients, one only can be used, or two or more can be usedtogether.

The shape (e.g., sphere, bar, needle, plate, amorphous, scale, spindle)and the particle size of these powders are not particularly limited.These powders may or may not be previously surface-treated by aconventionally known surface treatment such as fluorine compoundtreatment, silicone treatment, silicone resin treatment, pendanttreatment, saline coupling agent treatment, titanium coupling agenttreatment, lubricant treatment, N-acylated lysine treatment, polyacrylicacid treatment, metal soap treatment, amino acid treatment, lecithintreatment, inorganic compound treatment, plasma treatment andmechanochemical treatment. From among the above ingredients, one onlycan be used, or two or more can be used together.

(6) Specific Examples of Surfactant Anionic Surfactant:

Fatty acid soap, a-acyl sulfonate, alkyl sulfonate, alkylallylsulfonate, alkylnaphthalene sulfonate, alkyl sulfate, POE alkyl ethersulfate, alkylamide sulfate, alkyl phosphate, POE alkyl phosphate,alkylamide phosphate, alkyloylalkyl taurine salt, N-acylamino acid salt,POE alkyl ether carbonate, alkyl sulfosuccinate, sodiumalkylsulfoacetate, acylated hydrolyzed collagen peptide salt andperfluoroalkylphosphoric acid ester. From among the above ingredients,one only can be used, or two or more can be used together.

Cationic Surfactant:

Alkyltrimethylammonium chloride, stearyltrimethylammonium chloride,stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride,distearyldimethylammonium chloride, stearyldimethylbenzylammoniumchloride, behenyltrimethylammonium bromide, benzalkonium chloride,behenic acid amidopropyldimethyl hydroxypropylammonium chloride,diethylaminoethylamide stearate, dimethylaminoethylamide stearate,dimethylaminopropylamide stearate and lanolin derivative quaternaryammonium salt. From among the above ingredients, one only can be used,or two or more can be used together.

Amphoteric Surfactant:

Carboxybetaine type, amidobetaine type, sulfobetaine type,hydroxysulfobetaine type, amidosulfobetaine type, phosphobetaine type,aminocarboxylate type, imidazoline derivative type and amidoamine type.From among the above ingredients, one only can be used, or two or morecan be used together.

Nonionic Surfactant:

Propylene glycol fatty acid ester, glycerin fatty acid ester,polyglycerin fatty acid ester, sorbitan fatty acid ester, POE sorbitanfatty acid ester, POE sorbitol fatty acid ester, POE glycerin fatty acidester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castoroil, POE castor oil, POE-POP copolymer, POE-POP alkyl ether,polyether-modified silicone lauric acid alkanolamide, alkylamine oxideand hydrogenated soybean phospholipid. From among the above ingredients,one only can be used, or two or more can be used together.

Natural-Type Surfactant:

Lecithin, saponin and sugar-type surfactant. One ingredient only can beused, or two or more can be used together.

(7) Specific Examples of Polyhydric Alcohol and Sugar

Ethylene glycol, diethylene glycol, polyethylene glycol, propyleneglycol, dipropylene glycol, polypropylene glycol, glycerin, diglycerin,polyglycerin, 3-methyl-1,3-butanediol, 1,3-butylene glycol, sorbitol,mannitol, raffinose, erythritol, glucose, sucrose, fruit sugar, xylitol,lactose, maltose, maltitol, trehalose, alkylated trehalose, mixedisomerized sugar, sulfated trehalose and pullulan. Chemically modifiedproducts thereof can also be used. From among the above ingredients, oneonly can be used, or two or more can be used together.

(8) Specific Examples of Polymer Compound

Anionic polymer compounds such as acrylic acid ester/methacrylic acidester copolymer (PLUS-SIZE, produced by Sogokagaku K. K.), vinylacetate/crotonic acid copolymer (Resin 28-1310, produced by NSC), vinylacetate/crotonic acid/vinyl neodecanate copolymer (28-2930, produced byNSC), methyl vinyl ether maleic acid half ester (GANTREZ ES, produced byISP), T-butyl acrylate/ethyl acrylate/methacrylic acid copolymer(RUBIMER, produced by BASF), vinylpyrrolidone/vinyl acetate/vinylpropionate copolymer (RUBISCOL VAP, produced by BASF), vinylacetate/crotonic acid copolymer (RUBISET CA, produced by BASF), vinylacetate/crotonic acid/vinylpyrrolidone copolymer (RUBISET CAP, producedby BASF), vinylpyrrolidone/acrylate copolymer (RUBIFLEX, produced byBASF), acrylate/acrylamide copolymer (ULTRAHOLD, produced by BASF),vinyl acetate/butyl maleate-isobornyl acrylate copolymer (ADVANTAGE,produced by ISP), carboxy vinyl polymer (CARBOPOL, produced by BFGoodrich) and acrylic acid-alkyl methacrylate copolymer (PAMUREN,produced by BF Goodrich); amphoteric polymer compounds such as aceticacid amphoteric compound of dialkylaminoethyl methacrylate polymer(YUKAFORMER, produced by Mitsubishi Chemical) and octylacrylamideacrylate/hydroxypropyl acrylate/butylaminoethyl methacrylate copolymer(AMPHOMER, produced by NSC); cationic polymer compounds such asquaternized compound of vinylpyrrolidone/dimethylaminoethyl methacrylate(GAFQUAT, produced by ISP) and methyl vinyl imidazoliumchloride/vinylpyrrolidone copolymer (RUBICOTE, produced by BASF); andnonionic polymer compounds such as polyvinylpyrrolidone/vinyl acetatecopolymer (RUBISCOL VA, produced by BASF) andvinylpyrrolidone/dimethylaminoethyl methacrylate copolymer (COPOLYMERVC713, produced by ISP).

In addition, polymer compounds of natural origin, such as cellulose andderivatives thereof, calcium alginate, pullulan, agar, gelatin, tamarindseed polysaccharides, xanthane gum, carrageenan, high-methoxyl pectin,low-methoxyl pectin, guar gum, gum arabi, crystal cellulose, arabinogalactan, karaya gum, tragacanth gum, alginic acid, albumin, casein,cardrun, gellan gum and dextran, can also be suitably used. From amongthe above ingredients, one only can be used, or two or more can be usedtogether.

(9) Specific Examples of Biologically Active Ingredient

The biologically active ingredient may include substances which arecapable of imparting some biological activity to skin, when such asubstance is applied to the skin. Specific examples thereof may include:whitening ingredient, age resistor, ultraviolet protection, slimmingagent, skin tightening agent, antioxidant, hair restorer, hair growingagent, moisturizer, blood circulation accelerator, antibacterial agent,bactericide, desiccant, cooling agent, warming agent, vitamin compound,amino acid, wound healing accelerator, torpent, analgetic, cellactivator and enzyme ingredient.

Suitable examples of the ingredient to be blended therefor may include:angelica extract, avocado extract, hydrangea extract, althea extract,arnica extract, aloe extract, apricot extract, apricot core extract,ginkgo extract, fennel extract, turmeric extract, oolong tea extract,rose fruit extract, echinacea leaf extract, scutellaria root extract,phellodendron bark extract, goldthread extract, barley extract,hypericum extract, white nettle extract, watercress extract, orangeextract, sea salt, seaweed extract, hydrolyzed elastin, hydrolyzed wheatpowder, hydrolyzed silk, chamomile extract, carrot extract, artemisiacapillaris extract, glycyrrhiza extract, sabdariffa extract, pyracanthafortuneana fruit extract, cinchona extract, cucumber extract, guanosine,gardenia extract, sasa albo-marginata extract, sophora root extract,walnut extract, grapefruit extract, clematis extract, chlorella extract,mulberry bark extract, gentian extract, black tea extract, yeastextract, burdock extract, fermented rice bran extract, rice germ oil,comfrey extract, collagen, cowberry extract, asiasarum root extract,bupleurum falcatum root extract, umbilical cord extract, salvia extract,saponaria extract, bamboo grass extract, crataegus extract, zanthoxylumfruit extract, shiitake mushroom extract, rehmannia root extract,lithospermum root extract, perilla extract, linden extract, filipendulaextract, peony root extract, calamus rhizome extract, birch extract,horsetail extract, ivy extract, hawthorn extract, sambucus nigraextract, yarrow extract, peppermint extract, sage extract, mallowextract, cnidium rhizome extract, swertia herb extract, soy extract,jujube extract, wild thyme extract, green tea extract, clove extract,cogon extract, citrus unshiu peel extract, angelica root extract,calendula extract, peach seed extract, bitter orange extract, houttuyniaextract, tomato extract, natto extract, ginseng extract, garlic extract,wild rose extract, hibiscus sabdariffa flower extract, ophiopogon tuberextract, parsley extract, honey, witch hazel extract, pellitory extract,isodonis extract, matricaria extract, loquat extract, coltsfoot extract,butterbur scape extract, Poria cocos extract, butcher bloom extract,grape extract, propolis, luffa extract, safflower extract, peppermintextract, linden extract, peony extract, hop extract, pine extract, horsechestnut extract, skunk cabbage extract, sapindaceae extract, balm mintextract, peach extract, cornflower extract, eucalyptus extract,saxifrage extract, coix seed extract, mugwort extract, lavender extract,apple extract, lettuce extract, lemon extract, Chinese milk vetchextract, rose extract, rosemary extract, Roman chamomile extract androyal jelly extract, strawberry extract, piper longum extract, lotusleaf extract, pfaffia extract, star fruit extract or the like.

Other examples may include biopolymers such as deoxyribonucleic acid,mucopolysaccharide, sodium hyaluronate, sodium, elastin, chitin,chitosan and hydrolyzed eggshell membrane; moisture retentiveingredients such as amino acid, hydrolyzed peptide, sodium lactate,urea, sodium pyrrolidonecarboxylate, betaine, whey and trimethylglycine;oily ingredients such as sphingolipid, ceramide, phytosphingosine,cholesterol, cholesterol derivatives and phospholipid; anti-inflammatorysuch as E-aminocaproic acid, glycyrrhizic acid, -glycyrrhetic acid,lysozyme chloride, guaiazlene and hydrocortisone; vitamins such asvitamin A, vitamin B2, vitamin B6, vitamin D, vitamin E, calciumpantothenate, biotin and nicotinic acid amide; active ingredients suchas allantoin, diisopropylamine dichloroacetate and4-aminomethylcyclohexanecarboxylic acid; antioxidants such astocopherol, carotenoid, flavonoid, tannin, lignin and saponin; cellactivators such as a-hydroxy acid and hydroxy acid; blood circulationaccelerators such as y-orizanol and vitamin E derivatives; wound healingagents such as retinol and retinol derivatives; whitening agents such asalbumin, kojic acid, placenta extract, sulfur, ellagic acid, linoleicacid, tranexamic acid and glutathione; and hair growing agents such ascepharanthine, glycyrrhiza extract, capsicum tincture, hinokitiol,iodized garlic extract, pyridoxine hydrochloride, DL-a-tocopherol,DL-a-tocopheryl acetate, nicotinic acid, nicotinic acid derivatives,calcium pantothenate, D-pantothenyl alcohol, acetyl pantothenylethylether, biotin, allantoin, isopropylmethylphenol, estradiol, ethynylestradiol, capronium chloride, benzalkonium chloride, diphenhydraminehydrochloride, Takanal, camphor, salicylic acid, vanillylamide nonylate,vanillylamide nonanoate, pyroctone olamine, glyceryl pentadecanoate,L-menthol, mononitroguaiacol, resorcinol, y-aminobutyric acid,benzethonium chloride, mexiletine hydrochloride, auxin, female hormone,cantharis tincture, cyclosporine, zinc pyrithione, hydrocortisone,minoxidil, polyoxyethylene sorbitan monostearate, peppermint oil andSASANISHIKI extract. From among the above ingredients, one only can beused, or two or more can be used together.

(10) Specific Examples of Antioxidant

Sodium hydrogensulfite, sodium sulfite, erythorbic acid, sodiumerythorbate, dilauryl thiodipropionate, tocopherol, tolylbiguanide,nordihydroguaiaretic acid, parahydroxy anisole, butylhydroxy anisole,dibutylhydroxy toluene, ascorbyl stearate, ascorbyl palmitate, octylgallate, propyl gallate, carotenoid, flavonoid, tannin, lignin, saponinand plant extracts having antioxidant effect, such as apple extract andclove extract. From among the above ingredients, one only can be used,or two or more can be used together.

(11) Specific Examples of Solvent

Purified water, ethanol, lower alcohol, ethers, LPG, fluorocarbon,N-methylpyrrolidone, fluoroalcohol, volatile linear silicone and nextgeneration freon (such as fluorocarbon, chlorofluorocarbon, CFC). Fromamong the above ingredients, one only can be used, or two or more can beused together.

Example

Examples of this invention are described here, which identify the AGEinhibitory activity, or the like, of the compound obtained by thisinvention and which show that the scope of this invention is not limitedto its products and manufacturing methods.

(1) Method for Producing Extract of Cherry-Tree (Cherry Blossom, CherryLeaf):

Using cherry blossoms (referred to as “yaezakura” in Japanese, Prunuslannesiana Wils. cv. Sekiyama), an extraction was done with 30% (w/w)hydrous ethanol at 60 degree centigrade for one hour. After that,filtering and concentrating were done to obtain the cherry-blossomextract of a yield of 5 wt %. In the same way, cherry-leaf extract wasobtained of a yield of 4 wt %. Then, the cherry-blossom extract andcherry-leaf extract were applied, per Example 1 and Example 2,respectively.(2) Method for Separating the Active Substance from Cherry-BlossomExtract:

The search for substances held in the cherry-blossom extract is shown inFIG. 1. In other words, Example 1, the cherry-blossom extract (90.10grams) is suspended within the chromatography column (HP-20, MitsubishiChemical Corporation) to obtain eluted water (H2O) [of] (62.16 g,68.88%), eluted methanol (MeOH) [of] (28.21 g, 31.31%) and elutedacetone (Acetone) [of] (0.60 g, 0.67%).

Next, repeatedly separate and refine the eluted methanol (MeOH) of(26.00 g) within the normal-phased silica-gel chromatography column,within the reverse-phased ODS chromatography column and within the HPLC(high-performance liquid chromatography column) to isolate the knownthree-different phenylpropanoid glycosides:1-O-(E)-Caffeoyl-β-D-glucopyranoside (8.73 g, 9.6%),1-O-(E)-Coumaroyl-β-D-Glucopyranoside (22.1 mg, 0.0245%) and1-O-(E)-Cinnamoyl-β-D-glucopyranoside (21.0 mg, 0.0233%), as well as theknown four-different flavonoid glycosides: Kaempferol3-O-β-D-glucopyranoside (32.6 mg, 0.0361%), Quercetin3-O-β-D-glucopyranoside (86.0 mg, 0.0954%), Kaempferol3-O-(6″-malony)-β-D-glucopyranoside (55.3 mg, 0.0613%) and Quercetin3-O-(6″-malony)-β-D-glucopyranoside (66.2 mg, 0.0734%). (Examples 3 to 5and 6 to 9).

The known compounds, above are identified by comparison of theliterature values of 1H-NMR and ¹³C-NMR, Mass Spectral Database andoptical rotation. Also, each yield (w/w) of the isolated substances, asshown in FIG. 1, means an isolated yield of the cherry-blossom extract,Example 1.

(3) Effect Confirmation Test (Test 1: Effect of AGE ProductionInhibition)

To confirm the effect of the cherry-blossom extract and the cherry-leafextract on the formation of AGE, the following tests were done.

Put the sample solution (100 mL) of the concentration of cherry-blossomextract (Example 1) and cherry-leaf extract (Example 2), as shown in thechart, below, into a phosphate buffer solution (pH: 7.4, 900 mL)containing D-glucose (10%) and into bovine serum albumin (fraction 5.1%)and leave it out at a temperature of 60 degrees centigrade for two days.Dilute the reaction solution with distilled water until the intensity offluorescent light reaches about 500, thus determining its intensity(measurement wavelength: 370 nm, excitation wavelength: 440 nm). Also,the extract is to be diluted with distilled water, and the substance,after being dissolved in DMSO (dimethyl sulphoxide), is to be dilutedwith a phosphate buffer solution until DMSO concentration is of 1%. Theresult is shown in Chart 1, below. Each value in Chart 1 indicates anaverage value with standard error of the three examples. Asterisks (*,**) mean a significant difference (i.e. *: p_(<)0.05; **: p<0.01)between the processed samples and the unprocessed samples as determinedby Dunnett's Multiple Comparison Test.

CHART 1 AGE production inhibitory effect of cherry-blossom extract andits content substances Inhibition Ratio (%) IC50 1 (μg/mL) 3 10 30 100300 (μg/mL) Cherry-blossom extract −8.6 ± 0.5 −14.6 ± 0.7  −10.8 ± 0.4 −9.9 ± 0.6  15.1 ± 0.7** 42.6 ± 3.2** >300 Cherry-leaf extract −1.4 ±0.1 2.8 ± 0.1 7.0 ± 0.1 21.6 ± 0.4** 37.3 ± 1.4** 49.8 ± 0.4** >3001-O-(E)-Caffeoyl-β-D-glucopyranoside −3.3 ± 0.1 0.9 ± 0.1  10.7 ± 0.1**19.5 ± 0.3** 25.0 ± 0.3** 30.0 ± 0.4** >3001-O-(E)-Coumaroyl-β-D-glucopyranoside −3.2 ± 0.1  −8.2 ± 0.1**  −8.6 ±0.1** −8.9 ± 0.1** −3.7 ± 0.1** 11.6 ± 0.1** >3001-O-(E)-Cinnamoyl-β-D-glucopyranoside −8.4 ± 0.1 −10.4 ± 0.1  −10.9 ±0.4*  −7.8 ± 0.1  5.7 ± 0.1  23.3 ± 0.4** >300 Kaempferol3-O-β-D-glucopyranoside  −8.4 ± 0.1**  −9.1 ± 0.2** −2.0 ± 0.1  19.4 ±0.1** 45.0 ± 0.5** 80.3 ± 0.7** 102 Quercetin 3-O-β-D-glucopyranoside  8.5 ± 0.1**  6.5 ± 0.1*  27.6 ± 0.5** 49.8 ± 0.7** 74.2 ± 1.1** 100.8± 0.6**  30 Kaempferol 3-O-(6″-malony)-β-D- −5.0 ± 0.1 −8.5 ± 0.1  0.2 ±0.1 20.5 ± 0.3** 50.8 ± 0.4** 91.7 ± 1.7** 78 glucopyranoside Quercetin3-O-(6″-malony)-β-D- −10.4 ± 0.9  1.9 ± 0.1  20.4 ± 0.3** 43.7 ± 0.7**74.6 ± 0.7** 103.9 ± 3.6**  36 glucopyranoside Aminiguanidinehydrochloride — — 1.4 ± 0.1 18.1 ± 1.1  42.6 ± 1.7** 67.7 ± 1.6** 138Significant difference *p < 0.05, **p < 0.01

[Test 1: Result and Effect of Examples]

It is identified in Chart 1 that the cherry-blossom extract (Example 1)and cherry-leaf extract (Example 2), both in concentration of 100 to 300μg/mL, inhibit the production of AGE.

Also, as shown in Chart 1, the main polyphenol of cherry blossom, i.e.1-O-(E)-Caffeoyl-3-D-glucopyranoside (Example 3), in high concentrationof 300 μg/mL, shows a 30% inhibitory activity. The inhibitory activityof a related compound with less hydroxyl, i.e.1-O-(E)-Coumaroyl-β-D-Glucopyranoside (Example 4), and1-O-(E)-Cinnamoyl-β-D-glucopyranoside (Example 5) is reduced.Contrarily, the inhibitory activity of flavonoid glycoside is generallymuch, and the inhibitory activity of Quercetin 3-O-β-D-glucopyranoside(Example 7) and Quercetin 3-O-(6″-malony)-β-D-glucopyranoside (Example9) is much (IC50), more than twice as much as the inhibitory activity ofother compounds with Kaempferol as an aglycon. (IC50: half-maximal (50%)inhibitory concentration)

[Test 2-1: Inhibitory Activity Against Fibroblast Apoptosis Caused byCML-Collagen]

It is known that the main AGE factor, CML-collagen (Carboxymethyllysine-collagen), found in the skin, damages skin cells. Here, Test 2-1verifies the beneficial activity of cherry-blossom extract (Example 1)upon human fibroblast apoptosis caused by CML-collagen.

(1) Method of Preparing CML-Collagen

50 mg of 1 mM HCL was added to 25 mL of bovine skin-derived collagen anddissolved for two hours incubation at the temperature of 37 degreescentigrade, and was shaken as needed. Then, PBS (pH7.4 25 mL) was addedto sodium cyanoborohydride (1.42 g) and to glyoxylic acid (0.9 g) andmixed so as to react at the temperature of 37 degrees centigrade for 24hours. The reaction solution was then put into a dialysis cell of 10,000molecular mass for a dialysis-with-water test to be conducted for oneday and for a dialysis-with-PBS to be conducted for six days.CML-collagen (21 mg) was obtained by freeze-drying the dialysate.

(2) Fibroblast culture

Prior to the test below, a human diploid fibroblast (female/age40/normal skin-derived/PDL20) was cultured in the medium of fetal calfserum (FCS) with penicillin (100 units/mL) and streptomycin (100 mg/mL).

The fibroblast (7×104 cells) was suspended in the D-MEM mediumcontaining FCS (0.5%), penicillin (100 units/mL) and streptomycin (100mg/mL) to be seeded within 96-well plates (100 mL/well). After 24 hoursof culture, the dissolved CML-collagen solution (2 mg·mL) and the samplesolution (13 mL) were added to the medium and cultured for 24 hours.Collagen, instead of CML-collagen, was added to Normal group.

(4) Cell Survival Rate and Apoptosis Measurement

The rate of cell survival was measured by MIT Assay. The result is shownin Chart 2. At the same time, a microscopic test was done, of which theresult of formazan formation of fibroblast (×100) is shown in FIG. 2.Apoptosis was identified by measuring active caspase-3/7 by thefluorescence method, of which the result is shown in Chart 3, below.Each value of Charts 2 and 3 is indicated by the average value withstandard error of the six examples. Asterisks (*, **) mean a significantdifference (i.e. *: p<0.05; **: p<0.01) between the unprocessed samplesand Dunnett's multiple comparison test, and f represents p<0.05 byFisher's PLSD Test.

CHART 2 Effect of cherry-blossom extract against fibroblast cellsurvival rate (MTT Assay) Concentration Absorbance of (μg/mL) formazanformation (570 nm) Normal — 0.192 ± 0.007* Control — 0.172 ± 0.007Cherry-blossom 10 0.166 ± 0.002 extract 30 0.169 ± 0.002 100 0.175 ±0.002 300 0.174 ± 0.002 1000 0.185 ± 0.004 f N = 6, Average value ±standard error *p < 0.05 (Dunnett's Test) f p < 0.05 (Fisger's PLSDTest)

CHART 3 Effect of cherry-blossom against fibroblast apoptosis(caspase-3/7 Assay) Concentration Intensity of fluorescent light (μg/mL)(Excitation 480 nm/Detection 530 nm) Normal — 282 ± 9* Control — 331 ±12 Cherry- 10 301 ± 13 blossom 30 348 ± 11 extract 100 293 ± 16 f 300245 ± 12* 1000 139 ± 5** N = 6, Average value ± standard error, *p <0.05, **p < 0.01 (Dunnett's Test), f p < 0.05 (Fisher's PLSD Test)

[Test 2-1: Result and Effect of Examples] (1) Cell Survival Rate

According to Chart 2, above, the absorbent rate of the Control group ishigher than that of the Normal group, and the cell survival rate waslowered due to CML-collagen. As shown in FIG. 2, the microscopic imagesidentify the inhibition of formazan accumulation within the cell. Bythis situation, it is anticipated that fibroblast apoptosis is inducedby CML-collagen. Contrarily, in the group of which 1000 mg/mL of thecherry-blossom extract (Example 1) is added, the formazan formationsignificantly increased.

(2) Apoptosis Inhibition

As shown in Chart 3, above, caspase-3/7 activity of the Control groupincreases compared to that of the Normal group, and it is identifiedthat apoptosis is induced by the CML-collagen. Contrarily, in the Normalgroup of which the cherry-blossom extract (more than 100 mL) was added,caspase-3/7 activity decreased. When the volume of cherry-blossomextract was more than 300 mg/mL, caspase-3/7 activity was lower in theControl group than that in the Normal group, which suggests that thecherry-blossom extract possibly inhibits enzyme activity and enzymeexpression of caspase-3/7.

[Test 2-2: Inhibitory Activity of Cherry-Blossom Extract, andCherry-Leaf Extract and their Substances Against Fibroblast ApoptosisCaused by CML-Collagen]

Here, in Test 2-2, respecting the cherry-blossom extract (Example 1),and the cherry-leaf extract (Example 2) and the seven substancescontained in the cherry-blossom extract (Example 1):1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-Glucopyranosideand,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol3-O-3-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside and Quercetin3-O-(6″-malony)-β-D-glucopyranoside (Examples 3 to 9), the activityagainst fibroblast apoptosis, caused by CML-collagen, was identified inthe same way as in Test 2-1.

Also, in Test 2-2, PDL22-32 cells were used as the normal diploidfibroblast. As a target for comparison, aminoguanidine hydrochloridewhich is a medicine for the glycation inhibitor was examined in the sameway. The result is indicated in Chart 4, below. Each value of Chart 4indicates an average value with standard error of the five examples.Asterisks (*, **) mean a significant difference (i.e. *: p<0.05; **:p<0.01) between processed samples and unprocessed samples withCML-collagen, determined by Dunnett's Multiple Comparison Test.

CHART 4 Effect of Cherry-blossom/Cherry-leaf extract against fibroblastapoptosis (Caspase Assay) Inhibition Ratio (%) 1 (μg/mL) 3 10 30 100Cherry-blossom extract — 61.8 ± 2.6 77.1 ± 4.2*  Cherry-blossom leafextract (−13.2 ± 0.7) 62.7 ± 3.7 85.1 ± 3.1  (176.7 ± 13.7)** (−24.5 ±0.7) 46.9 ± 1.4 58.4 ± 1.9* (135.2 ± 3.1)** 1-O-(E)-Caffeoyl-β-D-glucopyranoside  26.2 ± 0.5* 37.6 ± 1.2  72.2 ±2.7* 1-O-(E)-Coumaroyl-β-D-glucopyranoside  17.2 ± 0.5  7.1 ± 0.2 51.1 ±1.9 1-O-(E)-Cinnamoyl-β-D-glucopyranoside −11.8 ± 0.3 19.7 ± 0.9 48.6 ±2.9 Kaempferol 3-O-β-D-glucopyranoside  −0.7 ± 0.1 27.9 ± 1.1 100.7 ±4.2  Quercetin 3-O-β-D-glucopyranoside  44.2 ± 1.5*  39.0 ± 1.1*  121.5± 5.4** Kaempferol 3-O-(6″-malony)-β-D-glucopyranoside −18.9 ± 0.6 −17.3± 0.6  10.5 ± 0.5 Quercetin 3-O-(6″-malony)-β-D-glucopyranoside  21.8 ±0.7 36.6 ± 1.4  98.4 ± 4.4* Aminoguanidine Hydrochloride 104.8 ± 34*  Significant difference *p < 0.05, **p < 0.01

[Test 2-2: Result and Effects of Examples]

As shown in Chart 4, the cherry-blossom extract (Example 1, 10 to 100mg/mL) lowered caspase-3/7 activity. From this result, it is suggestedthat the cherry-blossom extract (Example 1) inhibits apoptosis caused byCML-collagen. Among the contents, Quercetin 3-O-β-D-glucopyranoside(Example 7), shown to be the strongest substance in the AGE productioninhibitory Test 1, had the strongest inhibitory activity. Similarcompounds like Kaempferol 3-O-β-D-glucopyranoside (Example 6) andQuercetin 3-O-(6″-malony)-β-D-Glucopyranoside (Example 9) showed stronginhibitory activity. However, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside (Example 8) showed relatively weakactivity. As for cinnamic acids, Kaempferol 3-O-β-D-glucopyranoside,which contains the highest level of cinnamic acid, showed especiallystrong inhibitory activity.

As such, it is thought that the cherry blossom extract (Example 1), andthe cherry leaf extract (Example 2) and their active substances have AGEproduction inhibitory activity which inhibits fibroblast apoptosiscaused by CML-collagen. In light of the above results, it is seen thatthese active substances function as a breaker that degradesCML-collagen, which is an AGE. Thus, the AGE production inhibitor ofthis invention can be used to as an AGE decomposition agent or as an AGEremover.

[Test 3: Promoting Activity of Fibroblast Collagen Grating Formation]

When fibroblast is cultured in the presence of collagen solution,collagen grating formation can be identified. In a similar test, whenadding fibroblast that has been glycosylated by the intermediate productof glycation (glyoxal), it is known that the collagen grating formationis inhibited. Here, Test 3 showed the cherry-blossom extract inhibitsformation of the collagen grating of glycated human fibroblast.

(1) Glycation of Fibroblast

The infant-derived NB1 RGB (passage number 22) was suspended in theD-MEM complete media (1.86×104 cells/mL). 15 mL each of it was seededwithin each cell-culture dish of 14 cm in diameter. After 24 hours, eachsample and glyoxal were added until the molecular mass of each dishbecame 200 mM and incubate the dishes for five days.

(2) Collagen Grating Formation

Bovine skin-derived collagen (25 mg) was dissolved in 0.1% acetic acid(8.3 mL). Then, a 10-times concentration of Hanks solution (1.66 L) wasadded. Then, 0.1% acetic acid (3.5 mL) was added. 1M NaOH was added toneutralize the solution, which was sterilized by filteration to obtainthe collagen solution.

The mixed solution of collected fibroblast (5.4×104 cell) was suspendedin FCS (50 μm), and collagen (450 mL) was seeded within a 24-wellcell-culture plate and allowed to culture for 24 hours and 48 hours.Each culture was observed through a microscope and by the naked eye. Animage observed by the naked eye is shown in FIG. 3. A microscopic image(×100), [taken] after the 24-hour culture, is shown in FIG. 4. Amicroscopic image (×200) [taken] after the 48-hour culture, is shown inFIG. 5. For the purpose of comparison, aminoguanidin hydrochloride whichis an anti-glycation agent was also examined in the same way.

[Test 3: Result and Effects of Examples]

The result of the fibroblast and collagen solution culture, as shown inFIG. 3, is that collagen-grating formation was identified. In FIG. 3,the white hazy part shows the extension and projection of the fibroblastand collagen grating. The amount of collagen-grating formation of theControl group which was processed with glyoxal significantly decreasedcompared to that of Normal group. The well of fibroblast, processed with100 and 1000 mg/mL of cherry blossom-extract (Example 1) and glyoxalshowed an increase in the amount of grating formation. On the otherhand, the well of fibroblast processed with 100 mg/mL of guanidinehydrochloride, compared to the control group, showed no increase in theamount of grating formation. In FIG. 4, showing the microscopic imageafter 24 hours of culture, it was identified that the fibroblast of theNormal group and of the Control group and of guanidine hydrochloridewere spherically shaped. However, the well of 100 and 1000 mg/mL of thecherry blossom-extract (Example 1) identified the extension andprojection of the fibroblast. FIG. 5, the 200-times enlarged microscopicimage, after eight hours of culture, showed a few extensions of thefibroblast of the Control group. However, it was still weak compared tothat of the well of 100 and 1000 mg/mL of the cherry blossom-extract(Example 1).

[Test 4: AGE Production Inhibitory Activity within the Fibroblast]

Test 4 examined the activity of the cherry blossom-extract (Example 1)and of the 1-O-(E)-Caffeoyl-β-D-glucopyranoside (Example 3) against theproduction of AGE (carbon methyl lysine protein) within the fibroblast(human diploid fibroblast) which was processed with glyoxal.

Human diploid fibroblast (passage number 30) was suspended in a D-MEMcomplete culture media, and 20 mL each was seeded within a cell culturedish of 14 cm in diameter. After 48 hours of culture, samples of variousconcentrations and of 401M of glyoxal were added to 80%-confluent cellsand cultured for 5 days. After that, each sample was collected andcrushed. Then, protein 10 mg was examined by the Western Blotting methodusing an anti-AGE antibody to detect AGE. The result as shown in FIG. 6shows a detection of AGE by the anti-AGE antibody (HRP staining system).In FIG. 6, CaGlu means 1-O-(E)-Caffeoyl-β-D-glucopyranoside (Example 3).

[Test 4: Result and Effects of Examples]

As shown in FIG. 6, the detection of AGE using an anti-AGE antibodyidentified the amount of AGE that had been increased by the stimulationof glyoxal (comparison between Control and Non-control). Contrarily, thecherry blossom-extract (Example 1) of 10 mg/mL strongly inhibited theproduction of AGE. The effect of 100 mg/mL of the cherry-blossom extractwas reduced. Yet, inhibitory activity was still found.1-O-(E)-Caffeoyl-β-D-glucopyranoside (CaClu: Example 3) of 1 and 10mg/mL inhibited the production of AGE. From these results, it is nowclearly shown at the cellular level that cherry-blossom extract and itsmajor substances inhibits a production of AGE.

[Test 5: AGE Production Inhibitory Activity by the IntradermalAdministration of Glyoxal]

After continuously administrating the cherry-blossom extract (Example 1)in various concentrations, the AGE precursor, glyoxal, was intradermallyadministrated to observe its activity in the production of AGE. Based onthis observation, a recommended dose of the cherry-blossom extract(Example 1) was determined.

The solution (10, 50 and 100 mg/kg) of the cherry-blossom extract(Example 1) was administered to a mouse (ICR, male, 5-week-old) once aday for 10 days. After completing the administration, glyoxal (800 mM100 mL) was intradermally administered into the shaved back of themouse. After 21 hours, the last administration of cherry-blossom extract(Example 1) was done to the mouse. Then, after another three hours, themouse was immolated with an ether anesthesia, and theglyoxal-administered part was removed. The removed skin of the mouse wasthen crushed in 2 mL of lysis buffer (50 mM tris, 150 mM NaCl, 1% TritonX100, pH: 7.2) using Polytron Homogenizer, and the crushed material wascentrifugally separated (1000×g, 7 min). Then, the supernatant wascollected and refrigerated at the temperature of 20 degrees Centigradebelow zero.

After measuring the protein content of the frozen supernatant by the BCAmethod, the AGE content was measured using an ELISA kit (Trans Genic.Inc.) for measuring glucose-derived AGEs. In addition, the AGE wasdetected by the Western Blotting method. The result is shown in Chart 5,as well as in FIG. 7.

[Test 5: Result and Effects of Examples]

As shown in Chart 5 and in FIG. 7, the cherry-blossom extract(Example 1) indicated no dosage dependency. It was identified that 10 to100 mg/kg of the cherry-blossom extract, orally administration,inhibited the production of AGE. From this result, the recommendeddosage of the cherry blossom-extract (Example 1) is to be set between 10and 100 mg/day.

CHART 5 Effect of cherry-blossom extract against intradermal AGEproduction caused by glyoxal (Detected by ELISA) Dosage amount AGEInhibition ratio (mg/kg) (pg/mg protein) (%) Control — 53 ± 12 —Cherry-blossom 10 50 ± 15 5.7 extract 50 18 ± 17 66.0 100 30 ± 12 43.4Average value ± standard error (n = 6)

[Test 6: Dermal Cell Apoptosis Inhibitory Activity by IntradermalAdministration of CML-Collagen]

After the continuous administration of the cherry-blossom extract invarious concentrations, the CML-collagen was intradermally administratedto observe the activity against the apoptosis of dermal cells as well asthe fibroblast. The test was conducted in the following method.

The solution (10, 50 and 100 mg/kg) of cherry-blossom extract(Example 1) was administered to a mouse (ICR, male, 5-week-old) once aday for 10 days. After completing the administration, CML-collagen (100mg/100 mL) was intradermally administered into the shaved skin area ofthe mouse where the biauricular line of the occipital region of themouse crosses a midline. Collagen was administered to the Normal group.After 21 hours, the last administration of the cherry-blossom extract(Example 1) was done to the mouse. Then, after another three hours, themouse was immolated after being administrated an ether anesthesia. Thecollagen-administered part of the mouse was removed. The part removedfrom the mouse was immersed and fixed in a solution of 4%paraformadehyde, and the cut section was stained by TUNEL staining. Tocount the number of dermal cells, which were confirmed as being positiveby the TUNEL staining, the image of the cross section of the skin wastaken by a microscope (×400) and processed with color gradations frompurple to orange to identify the number of cells. To calculate the areaof the inner skin, a printed image of the cross section of the innerskin was measured by a digital planimeter, and the scale was multiplied.The result is shown in Chart 6, as well as in FIG. 8. In FIG. 8, thepart marked with a ▴ means TUNEL-positive cells.

[Test: Result and Effects of Examples]

By the administration of CML-collagen, the number of TUNEL-positivecells in the inner skin increased. To the contrary, the cherry-blossomextract (Example 1) decreased the number of TUNEL-positive cells in adosage-dependent manner. From this result, it was identified that thecherry-blossom extract (Example 1) inhibited fibroblast apoptosis of theskin caused by the CML-collagen, in vivo.

CHART 6 Effect of cherry-blossom extract against dermal cell apoptosiscaused by CML-collagen Dosage Number of TUNEL amount stained positiveInhibition ratio (mg/kg) cells (cells/mm²) (%) Normal — 27.2 ± 1.7 —Control —  36.4 ± 10.6 — Cherry-blossom extract 10 22.7 ± 5.4 149 5014.5 ± 2.9 238 100  8.8 ± 1.7* 300 Average value ± standard error (n =3), *p < 0.05

The following charts present examples of the compounds usingcherry-blossom extract (Example 1) as an AGE production inhibitor, orthe like. However the compounds shown below are not limited to justthese.

CHART 7 Blending example 1: Chewing gums Sugar 53.0 wt % Gum base 20.0Glucose 10.0 Starch syrup 16.0 Aroma chemical 0.5 AGE productioninhibitor, etc 0.5 100.0 wt %

CHART 8 Blending example 2: Gummies Reduction sugar 40.0 wt % Granulatedsugar 20.0 Glucose 20.0 Gelatine 4.7 Water 9.68 Yuzu juice (Citrusjunos) 4.0 Yuzu flavor 0.6 Pigment 0.5 AGE production inhibitor, etc 0.5100.0 wt %

CHART 9 Blending example 3: Candies Sugar 50.0 wt % Starch syrup 33.0Water 14.4 Organic acid 2.0 Aroma chemical 0.2 AGE production inhibitor,etc 0.4 100.0 wt %

CHART 10 Blending example 4: Yogurt (Hard type/Soft type) Milk 41.5 wt %Powdered skim milk 5.8 Sugar 8.0 Agar-agar 0.15 Gelatine 0.1 Lactic acidbacterium 0.005 AGE production inhibitor, etc 0.4 Aroma chemical Minuteamount Water Rest 100.0 wt %

CHART 11 Blending example 5: Soft drinks Fructose glucose solution 30.0wt % Emulsifying agent 0.5 AGE production inhibitor, etc 0.3 Aromachemical Appropriate amount Distilled water Rest 100.0 wt %

CHART 12 Blending example 6: Tablet-shaped sweets Sugar 76.4 wt %Glucose 19.0 Glycerine fatty acid ester 0.2 AGE production inhibitor,etc 0.5 Distilled water 3.9 100.0 wt %

CHART 13 Blending example 7: Soft capsules Brown rice germ oil 47.0 wt %Yuzu (Citrus junos) seed oil 40.0 Emulsifying agent 12.0 AGE productioninhibitor, etc 1.0 100.0 wt %

CHART 14 Blending example 8: Tablets Lactose 54.0 wt % CrystalineCellulose 30.0 Starch splitting product 10.0 Glycerin fatty acid ester5.0 AGE production inhibitor, etc 1.0 100.0 wt %

CHART 15 Blending example 8: Tablets Lactose 54.0 wt % CrystalineCellulose 30.0 Starch splitting product 10.0 Glycerin fatty acid ester5.0 AGE production inhibitor, etc 1.0 100.0 wt %

CHART 16 Blending example 10: Skin lotions Ethanol 5.0 wt % Glycerin 2.01,3-butylene glycol 2.0 Polyethylene oleyl ether 0.5 Sodium citrate 0.1Citric acid 0.1 AGE production inhibitor, etc 0.1 Distilled water Rest100.0 wt %

CHART 17 Blending example 11: Body gels Macadamia nut oil 2.0 wt % Octyldecyl myristate 10.0 Methylphenyl polysiloxane 5.0 Behenyl alcohol 3.0Stearic acid 3.0 Bathyl alcohol 1.0 Glycel monostearate 1.0 Tetra oleicacid polyoxyethylene 2.0 sorbit Hydrogenated soybean phosphatide 1.0Ceramide 0.1 Retinol palmitate 0.1 Preservative agent Appropriate amountCentella asiatica extract 1.0 AGE production inhibitor, etc 1.01,3-butylene glycol 5.0 Distilled water Rest 100.0 wt %

CHART 18 Blending example 12: Cosmetic emulsion Squalene 4.0 wt %Vaseline 2.5 Cetanol 2.0 Glycerin 2.0 Oleophilic glycerine monostearate1.0 Stearic acid 1.0 L-arginine 1.0 AGE production inhibitor 0.5Potassium hydroxide 0.1 Aroma chemical Minute amount Distilled waterRest 100.0 wt %

CHART 19 Blending example 13: Bath agent (liquid type) Propylene glycol50.0 wt % Ethanol 20.0 Sodium sulphate 5.0 AGE production inhibitor 0.5Lanoline 0.5 Avocado oil agent 0.5 Pigment 1.5 Aroma chemical 22.0 100.0wt %

Compounds 1 to 13, above, contain the cherry-blossom extract (Example1). Yet, they still may contain cherry-leaf extract (Example 2) or anyone of Examples 3 to 9:1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-Glucopyrano-side and,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol3-O-β-D-glucopyranoside, Quercetin 3-O-β-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside and Quercetin3-O-(6″-malony)-β-D-Glucopyranoside. It is also possible to use amixture of the above seven substances (Examples 3 to 9).

INDUSTRIAL APPLICABILITY

As described above, this invention makes it possible to inhibit theproduction of AGE and to prevent cells and intercellular matrix fromsuffering damage induced by AGE. From this result, this invention alsomakes it possible to effectively prevent and cure diabetes and diabeticcomplications, thus effectively preventing the skin from aging.

1-26. (canceled)
 27. The cherry-tree extract of this invention holds allcompounds, 1-O-(E)-Caffeoyl-β-D-glucopyranoside,1-O-(E)-Coumaroyl-β-D-glucopyranoside,1-O-(E)-Cinnamoyl-β-D-glucopyranoside, Kaempferol3-O-β-D-glucopyranoside, Quercetin 3-0,3-D-glucopyranoside, Kaempferol3-O-(6″-malony)-β-D-glucopyranoside, Quercetin3-O-(6″-malony)-β-D-glucopyranoside and said cherry-tree extract isobtained by a manufacturing method having the following three steps: (a)extracting one or both cherry blossom extract and the cherry leafextract with 20 to 50 wt % hydrous ethanol, (b) absorbing the extractobtained in step (a) by an absorbent, comprising porous synthetic resin,elute the extract in water and remove the eluted extract, and (c) afterstep (b) further eluting the eluted extract with a lower alcohol content(carbon numbers 1 to 5), and then concentrate the further elutedextract.
 28. An AGE production inhibitor comprising cherry-tree extractand/or its processed active substance according to claim
 27. 29. Anapoptosis inhibitor comprising cherry-tree extract and its processedsubstance as an active substance, according to claim 27, which inhibitsfibroblast apoptosis caused by the CML-collagen (Carboxymethyllysine-collagen) which is an AGE.
 30. An human fibroblast-collagengrating-formulation promoter comprising cherry-tree extract and/or itsprocessed substance as an active substance according to claim
 27. 31. AnAGE production inhibitor within the fibroblast comprising cherry-treeextract and/or its processed substance as an active substance accordingto claim
 27. 32. An agent, to prevent and cure the diabeticcomplications, comprising cherry-tree extract and/or its processedsubstance as an active substance according to claim
 27. 33. An agent, toprevent the skin from aging and to improve it, comprising cherry-treeextract and/or its processed substance as an active substance accordingto claim 27.